Microorganisms are able to switch from a planktonic, freeliving lifestyle in the environment to an aggregated, biofilm-associated state upon animal colonization. Across many organisms, this transition is associated with increased levels of cyclic dimeric GMP (c-di-GMP), an intracellular second messenger that coordinates the downregulation of motility genes and the upregulation of biofilm genes during the transition from the motile to the sessile lifestyle. Sessile, biofilm embedded bacterial colonies underlie chronic bacterial pathogenesis and bacteria within biofilms exhibit increased resistance to antibacterial treatments. Mechanisms regulating c-di- GMP turnover have been described, and the compound influences output phenotypes in various ways, including direct binding to proteins and to regulatory RNA. Previous work has relied on sophisticated systems in bacterial culture, eukaryotic cell culture, and animal models. However, little is known about the processes by which interaction with an intact animal stimulates a change in bacterial signal transduction. To understand the processes by which host-microbe interaction directs bacteria to adopt a sessile lifestyle, it is useful to study a model system in which individual stages can be dissected through genetics and imaging approaches. The light organ of the bobtail squid, Euprymna scolopes, is colonized exclusively by one bacterial species, Vibrio fischeri. Colonization is robust: it occurs within four hours via the native route and in the presence of host immune factors. Biofilm formation is required for initiating a successful colonization, and bacteria do not progress past this initial phase if they fail to aggregate in host mucus. We identified a bacterial mutant that dramatically increases aggregation behavior. The genetic lesion mapped to a hybrid histidine kinase, VF_A0360, and in addition to conferring increased aggregation the mutant also exhibited reduced flagellar motility and higher levels of c-di-GMP. Together, these data support the hypothesis that signal transduction through VF_A0360 coordinates the transition from the environmental state to the host-associated colonized state, and here we examine the following specific aims: (1) Identify genes regulated by the VF_A0360 histidine kinase and examine their dynamics during entry to and exit from squid colonization, and (2) Characterize the phosphorelay through which VF_A0360 regulates biofilm formation. By interrogating this signaling pathway we will gain insight into how bacterial signal transduction adjusts to the host environment during beneficial and pathogenic colonization.